A family of novel murine nuclear protein kinases (Homeodomain- Interacting Protein Kinase; HIPK1, HIPK2 and HIPK3) that interact with NK-homeoproteins and modulate their transcriptional activity were cloned both by yeast two-hybrid screen and cDNA library screening, and structures and functions of HIPKs were characterized. HIPKs differentially interact with known NK-homeoproteins and are nuclear proteins that phosphorylate NK-homeoproteins on threonine residues. HIPK2 greatly enhances the DNA binding activity of the NK-homeoprotein, independent of its phosphorylation state, but homeoprotein phosphorylation by HIPK2 affects the protein-protein interactions between homeoproteins. In cultured cells, interactions of HIPKs with NK-homeoproteins strongly potentiates repressor activities of NK- homeodomain transcription factors. Also, GAL4:HIPKs itself can function as potent transcriptional repressors when bound to DNA. Thus, these results suggest that HIPKs are co-repressors for homeodomain transcription factors and provide the first evidence that nuclear protein kinases (HIPKs) can function as co-repressors. Various GFP:HIPK2 fusion constructs and HIPK2-specific antibodies were generated in order to investigate the translocation of HIPKs into the nucleus and interactions of HIPKs with co-repressor complex, respectively. Also, further characterization of HIPK functions in relation to the functional specificity of NK-homeoproteins during development is underway.